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Regulation of microsomal prostaglandin E2 synthase by cyclopentenone prostaglandins in colon cancer cells [Elektronische Ressource] / von Yulyana Yudina

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100 pages
Regulation of Microsomal Prostaglandin E2 Synthase by Cyclopentenone Prostaglandins in Colon Cancer Cells Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften Vorgelegt beim Fachbereich für Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Yulyana Yudina aus Kharkov, Ukraine Frankfurt am Main (2005) (D 30) Vom Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann-Wolfgang-Goethe Universität als Dissertation angenommen. Dekan: Prof. Dr. Harald Schwalbe Gutachter: Prof. Dr. Dieter Steinhilber Prof. Dr. Dr. Jürgen Stein Datum der Disputation: 1 To my parents, 2 Table of contents: Abbrevations................................................................................................. 1 1 Introduction ............................................................................................... 4 1.1.1 Biological functions of prostaglandin E ........................................... 6 21.1.2 Prostaglandin E and cancer .............................................................. 7 21.2 Cyclopentenone prostaglandins ............................................................ 9 ∆12,141.2.1 15-deoxy- -PGJ .......................................................................... 10 21.
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Regulation of Microsomal Prostaglandin E2 Synthase by
Cyclopentenone Prostaglandins in Colon Cancer Cells




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften

Vorgelegt beim Fachbereich für
Chemische und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main


von
Yulyana Yudina
aus Kharkov, Ukraine




Frankfurt am Main (2005)
(D 30)




Vom Fachbereich Chemische und Pharmazeutische Wissenschaften der
Johann-Wolfgang-Goethe Universität als Dissertation angenommen.









Dekan: Prof. Dr. Harald Schwalbe
Gutachter: Prof. Dr. Dieter Steinhilber
Prof. Dr. Dr. Jürgen Stein

Datum der Disputation:


1





To my parents,
















2
Table of contents:

Abbrevations................................................................................................. 1
1 Introduction ............................................................................................... 4
1.1.1 Biological functions of prostaglandin E ........................................... 6 2
1.1.2 Prostaglandin E and cancer .............................................................. 7 2
1.2 Cyclopentenone prostaglandins ............................................................ 9
∆12,141.2.1 15-deoxy- -PGJ .......................................................................... 10 2
1.3 Enzymes involved in prostaglandin biosynthesis .............................. 12
1.3.1 Phospholipase A enzymes................................................................ 12 2
1.3.2 Cyclooxygenase enzymes .................................................................. 13
1.4 Prostaglandin E synthases ................................................................... 15
1.4.1 Cytosolic prostaglandin E synthase................................................. 15
1.4.2 Microsomal prostaglandin E synthase-1......................................... 16
1.4.3 Microsomal prostaglandin E synthase -2........................................ 17
2. Aim of the present investigation ........................................................... 20
3. Materials and Methods .......................................................................... 21
3.1.1 Chemicals ........................................................................................... 21
3.1.2 Antibodies 24
3.1.3 Mediums and Solutions..................................................................... 25
3.1.4 Cell lines ............................................................................................. 26
3.1.5 Instruments ........................................................................................ 27
3.1.6 Software.............................................................................................. 28
3.2.1 Cell culture......................................................................................... 28
3.2.2 Cell proliferation ............................................................................... 28
3.2.3 Cytotoxicity ........................................................................................ 29
3.2.4 Preparation of cellular fractions...................................................... 29
3.2.5 Analysis of mRNA levels using RT-PCR ........................................ 29
3.2.6 Immunoblot analysis ......................................................................... 30
3
3.2.7 PGES enzyme assay........................................................................... 31
3.2.8 Measurement of intracellular calcium level ................................... 31
3.2.9 Statistics.............................................................................................. 32
4. Results...................................................................................................... 33
4.1.1 Effect of 15d-PGJ on gene expression of the enzymes of 2
prostaglandin E2 synthesis ........................................................................ 33
4.1.2 15d-PGJ decreases mPGES-2 protein expression......................... 35 2
4.1.3 15d-PGJ reduces mPGES-2 enzyme activity................................. 38 2
4.2.1 Decreased expression of mPGES-2 in response to 15d-PGJ 2
treatment is PPAR γ independent.............................................................. 39
4.2.2 15d-PGJ does not affect mPGES-2 expression via cell surface 2
receptors ...................................................................................................... 43
4.2.3 The role of cyclopentenone ring structure in the down-regulation
of mPGES-2 expression.............................................................................. 45
4.2.4 Cyclopentenone prostaglandins induce oxidative stress................ 48
4.3.1 The effect of 15d-PGJ on cell growth............................................. 51 2
4.3.2 The implication of PGE formation in cell proliferation............... 52 2
5. Discussion ................................................................................................ 54
5.1 The role of mPGES-2 in colon cancer and its down-regulation by
anti-tumour cyclopentenone prostaglandin, 15d-PGJ . ......................... 54 2
5.2 The regulation of mPGES-2 by cyclopentenone prostaglandins is
independent of nuclear or membrane receptors activation, but may be
mediated by covalent binding of the cyclopentenone ring to cysteine
residues on signalling molecules or via a redox-dependent mechanism.
...................................................................................................................... 58
6. Summary ................................................................................................. 65
7. Zusammenfassung ............................... Fehler! Textmarke nicht definiert.
8. References ............................................................................................... 74
9. Appendix 90
4
10. Curriculum vitae .................................................................................. 91
11. Acknowledgements............................................................................... 93


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Abbrevations

12,1415d-PGJ 15-deoxy- ∆ -prostaglandin J2 2

AAArachidonic acid
ACFAberrantcryptfoci
AOM Azoxymethane
AP-2Activator protein-2
APC Adenomatous polyposis coli
ATM Ataxia-telangectasia mutated kinase
ATP Adenosine triphosphate
cDNA Complementary deoxyribonucleic acid
COX Cyclooxygenase
cPGES Cytosolic prostaglandin E synthase
cPLA Cytosolic phospholipase A
cAMP Cyclic adenosine monophosphate
CRE cAMP response element
CRTH2 Chemoattractant receptor–homologous molecule expressed on TH2 cells

DEPCDiethylpyrocarbonate
DMEM Dulbecco’s modified Eagle medium
dNTPs Deoxyribonucleotide triphosphates
DTTDithiothreitol

ECACC European Animal Cell Culture Collection
EGFR Epidermal growth factor receptor
Egr-1 Early growth response
EIA Enzyme immunoassay
EREndoplasmicreticulum
ERK1/2 Extracellular signal-regulated kinase1/2
1
FBS Foetal Bovine serum
FLAP 5-lipoxygenase-activating protein
Fura-2/AM Fura-2 acetoxymethyl ester

GAPDHGlyceraldehyde 3-phosphate dehydrogenase
GSH Glutathione
H-Ras Homologous to the oncogene of the Harvey rat sarcoma virus
HSA Human serum albumin
HSF-1 Heat shock transcription factor 1
HSP Heat shockprotein
IMN Indomethacin
IP3Inositol(1,4,5)-trisphosphate
iPLA Intracellular phospholipase A

JAK Janus protein kinase
JNK c-jun N-terminal kinase

LDHLactatedehydrogenase
LTLeukotriene

MAPEG Membrane Associated Proteins in Eicosanoid and Glutathione metabolism
MAPK Mitogen-activated protein kinases
MGST1-L1 Membrane-bound glutathione S-transferase 1-like-1
Min Multiple intestinal neoplasms
mPGES Microsomal prostaglandin E synthase
mRNA Messenger ribonucleic acid

NAC N-acetylcysteine
NF-IL-6 Nuclear factor for interleukin-6
NF- κB Nuclear factor-kappa B
NSAID Non-steroidal anti-inflammatory drugs
2

PBS Posphate buffered saline
PGProstaglandin
PGT Prostaglandintransporter
PKProtein kinase
PPAR Peroxisomeproliferator-activated receptor
PPREPPAR-response element

ROS Reactive oxygen species
RT-PCR Reverse transcription-polymerase chain reaction
RXR Retinoid X receptor

SAPK Stress-activated protein kinases
SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis
Sp1 Specificity protein 1
sPLA Secretory phospholipase A

TBS-T Tween Tris Base Saline
TxThromboxane

VEGFVascularendothelial growth factor



3
INTRODUCTION
1 Introduction


1.1 Prostaglandins

Prostaglandins, thromboxanes and leukotrienes, collectively referred to as
‘eicosanoids’, are the cyclooxygenase (COX) and lipooxygenase metabolites of arachidonic
acid (AA) (Fig. 1.). Discovery of eicosanoids (from Greek eicosa=twenty; for twenty carbon
fatty acid derivatives), was initiated in 1930. (Burr G. et al., 1930; Kurzrok R. et al., 1930;
Euler U. 1934). First, it was found that exclusion of fat from the diet of rats led to growth
retardation, reproductive disturbances, scaly skin, kidney lesions and excessive water
consumption, which led to the discovery of essential fatty acids. Second, a factor with fatty
acid properties and vasodepressor and smooth muscle-stimulating activity was identified that
was termed "prostaglandin." Bergström and Samuelsson linked these observations when they
elucidated the structures of the "classical" prostaglandins and demonstrated that they were
produced from an essential fatty acid, AA (Bergström S. et al., 1964).
All prostaglandins exhibit roughly the same structure as all are oxygenated fatty acids
composed of 20 carbon atoms and contain a cyclopentane ring, a C-13 > C-14 trans-double
bond, and a hydroxyl group at C-15 (Fig. 5, chapter 1.3). They were classified into types A to
I, dependent on the modifications of this cyclopentane ring. The abbreviations are commonly
followed by an index (for instance PGE ), which indicates the number of double bonds 2
present in the various side chains attached to the cyclopentane ring. Based on the number of
these double bonds, prostaglandins were classified into three series 1, 2, and 3.
Prostaglandins are formed by most cells of the body and act as autocrine and paracrine
lipid mediators, signalling at or immediately adjacent to their site of synthesis. They are not
only key mediators of inflammation and involved in apoptosis, cell differentiation and
oncogenesis, but also play critical physiologic roles in tissue homeostasis and function. For
example, gastric mucosal protective function, sleep induction and vascular smooth muscles
contraction and relaxation are all dependent upon these compounds. (Wallance J. 1992; Funk
C. 2001; Muller R. 2004). The actions of prostaglandins are summarized in Fig. 1.



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